SYNTHESIS OF NOVEL ACTIVITY BASED PROBES AND COMBINATORIAL PEPTIDE LIBRARIES TO PROFILE PROTEASES RESMI CHANDRASEKHARA PANICKER NATIONAL UNIVERSITY OF SINGAPORE 2007 SYNTHESIS OF NOVEL ACTIVITY BASED PROBES AND COMBINATORIAL PEPTIDE LIBRARIES TO PROFILE PROTEASES RESMI CHANDRASEKHARA PANICKER (M Sc - Mahatma Gandhi University, Kerala, India) A THESIS SUBMITTED FOR THE DOCTOR OF PHILOSOPHY DEGREE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS I wish to thank to my supervisor Associate Professor Yao Shao Qin for his novel ideas, patient guidance and invaluable suggestions during the course of my research I am grateful to my project partners Liau Minglee, Eunice, Haung Xuan and Wang Gang as well as other lab-mates Aparna, Souvik, Mahesh, Dawn, Wang Jun, Hong Yan, Elaine, Grace and Hu Yi for all their help during the course of the project Special thanks to my fellow-colleague plus life-partner Raja for his kind support and encouragement in all ways possible I appreciate the support of the laboratory staff from the NMR and the MS labs for providing me the necessary training and technical assistance I am also grateful to the National University of Singapore, for granting the research scholarship Last but not least, I would like to express my sincere thanks to my parents, grandmother, other family members and friends for their constant support and well wishes i TABLE OF CONTENTS Acknowledgements i Table of contents ii Summary vi List of tables viii List of figures ix List of schemes xii Abbreviations xiii Publications xvii Chapter Introduction 1.1 Proteomics 1.2 Conventional techniques for protein profiling 1.3 Activity based protein profiling 1.3.1 Activity-based probes 1.3.2 A few developments from our lab using activity 11 based/affinity based approaches 1.4 Combinatorial peptide libraries 13 1.4.1 Peptide Library on Solid Support 14 ii 1.4.2 Parallel synthesis 15 1.4.3 Split and mix synthesis 16 1.4.4 Reagent mixture synthesis 17 1.4.5 Positional scanning peptide libraries 17 1.5 Bioimaging 18 Chapter Design and Synthesis of activity-based probes targeting caspases 25 2.1 Introduction 25 2.1.1 Caspases 25 2.1.2 Mechanistic details of interaction of caspases with their substrates 27 2.2 Affinity tag approach to develop caspase probe for an in vitro proteomic Experiment 28 2.2.1 Design of the caspase-Cy3 probe 30 2.2.2 Chemical synthesis of the caspase-Cy3probe 33 2.2.3 Results and conclusions of the in vitro experiments 36 2.3 Activity based affinity probes for in vivo labeling of caspases 39 2.3.1 Design of cell permeable caspase probes 39 2.3.2 Chemical synthesis of cell permeable caspase probes 42 2.3.3 Results and conclusions of the in vivo labeling experiments 44 2.4 Experimental details of the synthesis of the caspase probes 47 iii Chapter Positional Scanning Peptide libraries of Fluorogenic substrates to map the substrate specificity of Proteases 58 3.1 Introduction 58 3.2 Design of our fluorogenic peptide library 60 3.3 Synthetic details of the ACC-library 62 3.4 Quality analysis of library 67 3.5 Fingerprinting experiments using ACC library 68 3.6 Data analysis and conclusions 68 3.7 ACC-azide library 71 3.8 Chemical synthesis of the azido-ACC-peptides 71 3.9 Experimental details of Syntheses 72 3.9.1 Experimental details of synthesis of ACC-Positional scanning library 72 3.9.2 Experimental details of synthesis of azido-ACC peptides 81 Chapter Fingerprinting of metalloproteases and cysteine proteases using positional scanning peptide libraries 84 4.1 Affinity based fingerprinting of metalloproteases using a positional scanning inhibitor library of peptidyl hydroxamates 84 4.1.1 Synthesis of the peptidyl hydroxamate library 86 4.1.2 Gel-based inhibition experiments using the peptidyl hydroxamate library 87 4.2 Activity based fingerprinting of cysteine proteases using a positional scanning library of peptide vinyl sulfone probes 89 iv 4.2.1 Synthesis of the vinyl sulfone library 91 4.2.2 Results and conclusions from the labeling experiments 93 4.3 Experimental details of the synthesis 94 4.3.1 Experimental details of the synthesis of the peptide hydroxamate library 94 4.3.2 Experimental details of the synthesis of the vinyl sulfone library 98 Chapter Synthesis of Molecular probes for potential bioimaging experiments 107 5.1 Introduction 107 5.2 Design of the NTA probes 108 5.3 Chemical synthesis of the probes 109 5.4 Results and conclusions of the labeling experiments 111 5.5 Experimental details of the synthesis of NTA probes 113 Chapter References 114 Chapter Appendices 129 Capase-Fluorescein probe (16) – 1H NMR 129 Capase-Fluorescein probe (16) – ESI-MS 130 Capase-Biotin probe (18) – 1H NMR 131 Capase-Biotin probe (18) – ESI-MS 132 AVLQ-ACC-Lys(N3) – ESI-MS 133 NTA-TMR (57) - ESI-MS 134 NTA-FL (59) - ESI-MS 135 v SUMMARY The work presented in this thesis focus mainly on two areas (i) designing specific probes to target a particular class of proteases using activity based affinity tag approach (ii) collection of substrate specificity/binding data of proteases using positional scanning peptide libraries Chapter of the thesis present our efforts towards the design and synthesis of small molecule probes to target caspases, enzymes which play a key mediating role in apoptosis or programmed cell death At first, we synthesized a fluoromethyl ketone containing activity-based probe that specifically target caspases in an in vitro proteomic experiment Later on, we extended this approach to the in vivo labeling of caspases in apoptotic HeLa cells by the use of modified probes which are cell permeable The attractive feature of our strategy is that it allows for the large scale identification of novel enzyme-associating proteins Chapter 3, and mainly focus on the synthesis of positional scanning combinatorial libraries of peptide substrates/inhibitors to profile proteases These works concentrate on the studies of the substrate specificity or “fingerprinting” of various classes of proteases For example, a positional scanning library of 7-amino-4 carbamoylmethylcoumarin (ACC) conjugated peptides were synthesized and assayed against different classes of proteases The substrate specificity profiles of various classes of proteases were successfully obtained using this library Other efforts include the activity-based profiling of cysteine proteases using a twenty member library of vinyl sulfone-containing peptides with varying P1 position and the synthesis of a positional vi scanning combinatorial library of peptidyl hydroxamates to investigate the substrate specificity of metalloproteases at the P2-P4 positions A brief attempt for bioimaging using small molecular probes also has been done as illustrated in chapter Selected NMR and MS spectra are listed in the Appendices vii LIST OF TABLES Table Proteins identified by mass spectrometry 47 Table Optimization of conditions for ACC coupling 65 Table ESI-MS data of the ACC-conjugated peptide azides 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Caspase-Biotin probe (18) – ESI-MS 132 AVLQ-ACC-Lys(N3) – ESI-MS 133 NTA-TMR (57) 134 NTA-FL (59) 135 ...SYNTHESIS OF NOVEL ACTIVITY BASED PROBES AND COMBINATORIAL PEPTIDE LIBRARIES TO PROFILE PROTEASES RESMI CHANDRASEKHARA PANICKER (M Sc - Mahatma Gandhi University, Kerala,... mainly focus on the synthesis of positional scanning combinatorial libraries of peptide substrates/inhibitors to profile proteases These works concentrate on the studies of the substrate specificity... representation of activity- based profiling strategy 1.3.1 Activity- based probes Activity- based probes are designed based on their potential application in proteomics The design of these probes depends